Gamma-irradiated rotavirus: A possible whole virus inactivated vaccine

Figures

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  Fig 1. Inactivation curves of RV and SFV following exposure to γ-irradiation. SFV and RV samples were exposed to increased doses of γ-irradiation on dry-ice, and the reduction in virus titre was determined by (A) plaque forming assay for SFV, or (B) fluorescent focus assay for RV. All samples tested in triplicate and data presented as mean ± SEM.

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  Fig 2. Sterility testing of 50 kGy γ-irradiated RV. Monolayers of MA104 cells were incubated for 24h with live RV or γ-RV at 4 x 105 FFU-equivalent/well. Culture supernatant was harvested and used to infect new MA104 cell monolayers, and previously infected monolayers stained for RV infection by FFA. DAPI channel (blue) indicates cell nuclei, and FITC channel (green) indicates RV infection. Stained monolayers visualised using Nikon TiE inverted fluorescence microscope. Scale bar = 100 μm. Images representative of 5 replicates per sample for each passage.

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  Fig 3. The ability to detect very low FFU of live RV. Monolayers of MA104 cells were incubated for 24h with 0.2 to 2000 FFU live RV. Uninfected MA104 cells were used as a negative control. Culture supernatant was used to infect new MA104 monolayers, and previously infected monolayers were visualised by FFA. DAPI (blue) indicates cell nuclei and FITC (green) indicates RV infection. Stained monolayers were visualised using Nikon TiE inverted fluorescent microscope. Scale bar = 100μm. Images representative of 3 replicates per MOI for each passage.

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  Fig 4. RV-specific antibody responses. Mice were primed twice with live RV or γ-RV, 2 weeks apart. Serum samples harvested on Day 14 post-2nd priming and analysed for RV-specific IgG using ELISA. (A) Serial dilutions of serum samples and absorbance readings at 450/620 nm for total IgG. (B) IgG titres in primed groups calculated relative to cut-off value (dotted line), determined using OD values of serum from control mice. Data presented as mean ± SEM (n = 6), and analysed by unpaired t-test (n.s., not significant) (C) Absorbance at 450/620nm for total IgG, IgG1, and IgG2c in serum at 1:200 dilution by ELISA. Data presented as mean ± SEM (n = 4), analysed by One-Way ANOVA (****, p < 0.0001).

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  Fig 5. Neutralising antibody responses induced by γ-RV. Mice were primed with live RV or γ-RV twice, 2 weeks apart. Serum samples harvested on Day 21 post-2nd priming, and neutralising ability of immune serum determined by in vitro neutralisation assay. (A) FFU/well determined following incubation of MA104 cells with sera treated-RV at MOI 0.005. RV treated with serial dilutions of HI control or immune sera. PBS-treated RV used to indicate the baseline level of infection. Data presented as mean ± SEM (n = 2), and analysed by One-Way ANOVA (****, p < 0.0001 compared to naïve control sera for each dilution. #, p < 0.05, when directly comparing immune sera groups). (B) Representative fluorescence images of RV infection after treatment with live or γ-RV sera at 1:1280 dilution. DAPI channel (blue) indicates cell nuclei, and FITC channel (green) indicates RV infection. Scale bar = 100 μm.